Reciprocal changes in amino acid metabolism in mammary gland and liver of the lactating rat on starvation and refeeding as indicated by the tissue accumulation of alpha-amino[1-14C]isobutyrate.

Measurements of the tissue accumulation in vivo and in vitro by hepatocytes and mammary-gland acini of alpha-amino[1-14C]isobutyrate ([1-14C]AIB) were compared in virgin and lactating rats. The results indicate the existence of a reciprocal relationship between mammary gland and liver for AIB accumulation that is dependent on the lactational and the nutritional state of the rat. This suggests that amino acids are preferentially directed to the mammary gland during active lactation.     

A static model to analyze carbon and nitrogen partitioning in the mammary gland of lactating sows

Quantitative estimates of mammary nutrient inputs, outputs and metabolism in sows are scarce, despite being critical elements to identify parameters controlling milk synthesis central for the feeding of lactating sows. The objective of this study was to quantify the mammary gland input and output of nutrients as well as the intramammary partitioning of carbon and nitrogen with the purpose to identify mechanisms controlling mammary nutrient inputs, metabolism and milk production in lactating sows. A data set was assembled by integration of results from four studies. The data set included data on litter performance, mammary arterial-venous concentration differences (AV-difference) of energy metabolites and amino acids, and the contents of lactose, fat and amino acids in milk. Milk yield was estimated based on average litter size and litter gain, and mammary plasma flow (MPF) was estimated using the sum of phenylalanine and tyrosine as internal flow markers. The yield and composition of milk were used to estimate mammary nutrient output in milk, and MPF and AV-difference were used to estimate net mammary input of carbon and nitrogen and output of CO₂. Carbon and nitrogen used for the synthesis of lactose, fat and protein in milk and CO₂-yielding processes were represented in a static nutrient partitioning model. The origin of mammary CO₂ output was calculated using theoretical estimates of carbon released in processes supporting mammary synthesis of de novo fat, protein and lactose in milk, mammary tissue protein turnover and transport of glucose and amino acids. Results indicated that total input of carbon from glucose and lactate was partitioned into lactose (36%), fat (31%) and CO₂-yielding processes (34%). Theoretical CO₂ estimates indicated that de novo fat synthesis, milk protein synthesis and mammary tissue protein turnover were the main processes related to mammary CO₂ production. More than 90% of mammary gland amino acid input was used for milk protein. The quadratic relationship between AV-difference and mammary input of essential amino acids indicated that both changes in AV-difference and MPF contributed to the regulation of mammary input of essential amino acids. The impact of the arterial supply of amino acids on mammary input may be greater for the branched-chain amino acids, arginine and phenylalanine than for other essential amino acids. In conclusion, relationships between input and output parameters indicate that AV-difference and MPF regulate mammary nutrient input to match the supply and demand of nutrients for the mammary gland.     

The regulation of Na(+)-dependent anionic amino acid transport by the rat mammary gland.

The regulation of anionic amino acid transport, using radiolabelled D-aspartate as a tracer, by rat mammary tissue explants has been examined. Na(+)-dependent D-aspartate uptake by mammary tissue increased between late pregnancy and early lactation and again at peak lactation but thereafter declined during late lactation. In contrast, the Na(+)-independent component of D-aspartate uptake by mammary explants did not change significantly with the physiological state of the donor animals. Premature weaning of rats during peak lactation markedly decreased Na(+)-dependent D-aspartate uptake by mammary tissue. In addition, premature weaning also reduced the effect of reversing the trans-membrane Na(+)-gradient on the fractional loss of D-aspartate from mammary tissue explants. Unilateral weaning of rats during peak lactation revealed that milk accumulation per se reduced the Na(+)-dependent moiety of D-aspartate uptake by mammary tissue suggesting that the transport of anionic amino acids is regulated to match supply with demand. Treating lactating rats with bromocryptine reduced D-aspartate uptake by mammary tissue explants suggesting that the transport of anionic amino acids by the rat mammary gland is regulated by prolactin.     

A study of l-leucine, l-phenylalanine and l-alanine transport in the perfused rat mammary gland: possible involvement of LAT1 and LAT2

The transport of l-leucine, l-phenylalanine and l-alanine by the perfused lactating rat mammary gland has been examined using a rapid, paired-tracer dilution technique. The clearances of all three amino acids by the mammary gland consisted of a rising phase followed by a rapid fall-off, respectively, reflecting influx and efflux of the radiotracers. The peak clearance of l-leucine was inhibited by BCH (65%) and d-leucine (58%) but not by l-proline. The inhibition of l-leucine clearance by BCH and d-leucine was not additive. l-leucine inhibited the peak clearance of radiolabelled l-leucine by 78%. BCH also inhibited the peak clearance of l-phenylalanine (66%) and l-alanine (33%) by the perfused mammary gland. Lactating rat mammary tissue was found to express both LAT1 and LAT2 mRNA. The results suggest that system L is situated in the basolateral aspect of the lactating rat mammary epithelium and thus probably plays a central role in neutral amino acid uptake from blood. The finding that l-alanine uptake by the gland was inhibited by BCH suggests that LAT2 may make a significant contribution to neutral amino acid uptake by the mammary epithelium.     

Expression of kappa-casein in normal and neoplastic rat mammary gland is under the control of prolactin

An 820-nucleotide-long cDNA clone for the kappa-casein (the casein micelle-stabilizing protein) from rat mammary gland was isolated, and its nucleotide sequence was determined. The deduced amino acid sequence from the nucleotide sequence revealed a signal peptide, 21 amino acids long, and a mature protein of 157 amino acids. The signal peptide of rat kappa-casein was highly homologous to that of the precursor to ovine kappa-casein. However, little homology was apparent when the mature kappa-casein protein sequences from ovine or bovine sources were compared with rat kappa-casein. The kappa-casein mRNA content of the mammary tissue was found to increase during its functional differentiation. Prolactin appears to modulate the production of kappa-casein mRNA. Mammary glands of virgin females had no detectable kappa-casein mRNA; however, a marked induction of kappa-casein mRNA was obtained by intravenous infusion of prolactin. Mammary carcinomas did not follow the same pattern. 7,12-Dimethylbenz[a]anthracene-induced mammary carcinomas had normally low levels of kappa-casein mRNA, but intravenous prolactin infusion increased the levels by 2-fold. The MTW9 mammary carcinoma that grows only in the presence of high levels of mammotropic hormones had kappa-casein mRNA content equivalent to that in 10-day lactating rat mammary gland. Continuous venous infusion of prolactin to MTW9 mammary carcinoma did not modify the kappa-casein mRNA levels. Nitrosomethylurea-induced mammary carcinomas had no detectable kappa-casein mRNA, and intravenous prolactin infusion was unable to induce it.     

Studies on the mode of uptake of blood triglycerides by the mammary gland of the lactating goat. The uptake and incorporation into milk fat and mammary lymph of labelled glycerol, fatty acids and triglycerides

1. The mode of uptake of the precursors of milk fat by the mammary gland of the lactating goat has been examined by infusing radioactive fatty acids, glucerol or doubly labelled triglycerides into the mammary artery or jugular vein of animals surgically prepared to permit samples of arterial and venous blood to be withdrawn without disturbance to the animal. 2. Acetate was taken up by the mammary gland and incorporated into milk fat. The decrease in the specific radioactivity of blood acetate across the gland was evidence of acetate production, but there was no significant release of labelled lipid from the mammary gland. 3. When labelled long-chain fatty acids or glycerol were infused into the lactating goat, there was extensive transfer of radioactivity into milk in spite of the absence of net uptake of substrate by the mammary gland. The decrease in the specific radioactivity of each substrate across the mammary gland, however, showed that both fatty acids and glycerol were simultaneously taken up and released by mammary tissue. 4. The infusion of chylomicra and triglyceride emulsions labelled with (3)H and (14)C revealed that both glycerol and fatty acids were released during triglyceride uptake by mammary tissue. Changes in the (3)H/(14)C ratio during the transfer of triglyceride from blood into milk showed that at least 80% of the triglyceride was hydrolysed during uptake, but the potential re-utilization of both products of hydrolysis for triglyceride synthesis in mammary tissue implied that only a minimum value could be obtained from the change in the ratio. 5. The time-course of the transfer of (3)H and (14)C into milk and lymph were closely similar after the infusion of [2-(3)H]glycerol tri[1-(14)C]oleate or of a mixture of [2-(3)H]glycerol and [1-(14)C]oleate. 6. The results were consistent with the hypothesis that plasma triglycerides are extensively or completely hydrolysed during mammary uptake.     

Uptake and metabolism of tryptophan by the isolated perfused guinea-pig mammary gland.

Tryptophan uptake by the isolated perfused lactating guinea-pig mammary gland was 46.5+/-4.6 mug/h per g. Results of absorption studies and the use of [methylene-14C]tryptophan suggest that tryptophan is one of the group of amino acids that are transferred almost quantitatively from blood plasma to milk protein.     

Cloning and analysis of cDNA encoding bovine butyrophilin, an apical glycoprotein expressed in mammary tissue and secreted in association with the milk-fat globule membrane during lactation

Butyrophilin is a glycoprotein expressed on the apical surfaces of secretory cells in lactating mammary tissue, which may function in the secretion of milk-fat droplets (Franke, W. W., Heid, H. W., Grund, C., Winter, S., Freudenstein, C., Schmid, E., Jarasch, E.-D., and Keenan, T. W. (1981) J. Cell Biol. 89, 485-494). A cDNA clone encoding bovine butyrophilin was isolated and the primary structure of the protein deduced from the DNA sequence. Bovine butyrophilin contains 526 amino acids with a putative signal peptide of 26 amino acids. Hydropathy analysis predicts the existence of a single membrane-spanning region with the amino terminus facing the exoplasmic space. Butyrophilin cDNA hybridized to mRNA of 2.9 kilobases in bovine mammary tissue taken from pregnant or lactating animals, but specific mRNA was not detected in a 2-year-old virgin cow. The amount of message was maximal in lactating tissue. Butyrophilin mRNA could not be detected in bovine heart, intestine, kidney, liver, ovary, or uterus. A search of the GenBank and EMBL data banks showed closest homology was between the C termini of butyrophilin and "ret finger protein" (Takahashi, M., Inaguma, Y., Hiai, H., and Hirose, F. (1988) Mol. Cell. Biol. 8, 1853-1856). The ret-finger protein gene is expressed in a variety of tumor cell lines, mouse testis and embryonic tissue, which like the mammary gland undergo periods of rapid cell division and development. The possible significance of this homology and the possible function of butyrophilin in milk-lipid secretion are discussed.     

Involvement of gamma-glutamyltransferase in amino-acid uptake by the lactating mammary gland of the rat.

1. Arteriovenous differences of amino acids across the lactating mammary gland have been measured in normal rats and in rats injected with serine--borate (an inhibitor of gamma-glutamyltransferase). 2. Comparison of the arteriovenous differences show that gamma-glutamyltransferase is involved in amino-acid uptake by the gland. 3. Reduced-glutathione content of isolated acini incubated with high concentrations of amino acids was lower than that of the controls. 4. High concentrations of amino acids had no effect on reduced-glutathione content of isolated acini when serine--borate was added to the incubation medium. 5. The findings provide evidence for the functioning of the gamma-glutamyl cycle in the lactating mammary gland in vivo.     

Metabolism of aminoacyl-p-nitroanilides by rat mammary tissue

We have examined the metabolism of aminoacyl-p-nitroanilides by rat mammary tissue isolated from rats during late pregnancy, peak lactation and late lactation. The rate of hydrolysis depended upon the chemical nature of the aminoacyl-p-nitroanilide compound and the physiological state of the donor animals. Thus, mammary tissue isolated from rats during late pregnancy and peak lactation hydrolysed aminoacyl-p-nitroanilides in the order L-met-p-nitroanilide=L-leu-p-nitroanilide>L-lys-p-nitroanilide>gamma- glu-p-nitroanilide. The order of activity was the same for mammary tissue taken from rats during late lactation except that L-lys-p-nitroanilide was hydrolysed at the same rate as the neutral aminoacyl-p-nitroanilides. Mammary tissue from peak lactating rats also hydrolysed alpha-L-glu-p-nitroanilide and alpha-L-asp-p-nitroanilide but to a lesser extent than the other compounds tested. The anionic aminoacyl-p-nitroanilides were able to trans-stimulate D-aspartate efflux from mammary tissue explants and the perfused mammary gland via the high-affinity anionic amino acid carrier. The clearance of gly-L-phe by the perfused mammary gland was markedly inhibited by L-phe. The results suggest that mammary tissue expresses a variety of dipeptidases at the basolateral aspect of the mammary epithelium which are capable of hydrolysing peptides extracellularly. These enzymes may be important for providing amino acids for milk protein synthesis and/or inactivating signal peptides.     

Chylomicron margination, lipolysis, and vitamin a uptake in the lactating rat mammary gland: implications for milk retinoid content

We have reported previously that the concentration of vitamin A (VA) in the milk of lactating rats varies with dietary VA intake, even when plasma retinol concentration is unaffected. In the current study, we investigated the role of lipolysis in the uptake of chylomicron (CM) VA into mammary tissue of lactating rats and estimated the proportion of CM-VA that is associated with the mammary gland during CM clearance. Chylomicrons containing [(3)H]VA, mainly as retinyl esters, were prepared in donor rats and administered intravenously to lactating recipient rats. Chylomicron VA rapidly disappeared from plasma and appeared in mammary tissue (maximum within 2-3 mins), followed by a decline. Concomitantly, uptake by liver increased continuously, reaching a plateau within 20-30 mins. Active lipolysis in mammary tissue was necessary for rapid VA uptake, as significantly less CM-VA was recovered in mammary tissue of postlactating rats than of lactating rats, after heparin treatment in lactating rats, or after injection of preformed CM remnants in lactating rats. [(3)H]Vitamin A uptake by mammary tissue increased linearly with CM-VA dose over a 150-fold dose range (R(2) = 0.972, P = 0.0001), suggesting a high capacity for uptake and apparent first-order assimilation of CM-VA during CM remnant formation in situ. Model-based compartmental analysis using WinSAAM predicted that approximately 42% of CM-VA marginated, that is, were temporarily removed, from plasma to the mammary glands during lipolysis and that a total of 3.8% of CM-VA was transferred to mammary tissue. The model-predicted t(1/2) for CM remnants was 3.04 mins. The metabolism of CM-VA in the lactating mammary gland, in proportion to VA absorption and CM-VA contents, may explain how milk VA concentration varies even when plasma retinol levels are unchanged. The mechanism of CM margination and mammary gland uptake described here for VA may be similar for other lipophilic substances.     

Heterogeneity of transport systems for L-glutamine in mouse mammary gland

The characteristics of the transport systems of L-glutamine in lactating mouse mammary gland have been studied. L-glutamine uptake was mediated by three Na+-dependent and one Na+-independent systems. The 2-(methylamino)isobutyric acid-sensitive component of Na+-dependent uptake exhibited the usual characteristics of system A. The other two Na+-dependent systems, which we have named BCI(-)-dependent and BCl(-)-independent, are the new systems identified. These are broad specificity systems and were discriminated on the basis of inhibition analysis, Cl- dependency and the effect of preloading mammary tissue with amino acids. While L-aspargine inhibited the uptake of L-glutamine via both these broad specificity systems, L-homoserine inhibited the uptake of L-glutamine via only BCl(-)-dependent system. The uptake of L-glutamine via the BCl(-)-independent system was upregulated by preloading mammary tissue with L-serine, while BCl(-)-dependent system was unaffected. The Na+-independent uptake of L-glutamine was inhibited by 2-aminobicyclo-(2,2,1)heptane carboxylic acid and other neutral amino acids, and identified as the system L.     

Uptake of free plasma amino acids by the lactating cow''s udder and amino acid composition of udder lymph

1. Total α-amino N and the amounts of 24 ninhydrin-positive substances were determined in several samples of plasma and lymph from the cow's udder. The arteriovenous differences of these substances across the mammary glands were measured in several experiments performed on lactating cows and in one experiment on a `dry' cow. Udder lymph obtained from live lactating cows by a lymph fistula and taken after killing lactating cows was analysed. 2. The concentrations of the individual free amino acids in udder lymph obtained from the live cow were similar to those found in cow's plasma. The concentrations of many amino acids in udder lymph taken immediately after death were two- to four-fold higher than those of the corresponding amino acids in udder lymph obtained from the live cow. 3. Most amino acids of the blood showed a considerable decrease in concentration by passage across the lactating mammary gland. Ornithine, a non-casein amino acid, showed arteriovenous differences of up to 60% of the arterial plasma concentration. No substantial amino acid uptake by the udder could be demonstrated in the experiment on the non-lactating cow. 4. The arteriovenous differences obtained for arginine, glutamine, isoleucine, leucine, lysine, valine, threonine and histidine were probably large enough to provide all the respective amino acid residues in milk protein. 5. The uptake of aspartic acid, asparagine, glutamic acid, serine and proline by the lactating cow's udder was not sufficient to account for all these respective amino acid residues found in milk protein.     

Cellular uptake of taurine by lactating porcine mammary tissue

Milk taurine plays a critical role in neonatal development. Taurine uptake in lactating sow mammary tissue has not been characterized previously. The kinetic properties, ion dependence and substrate specificity of taurine uptake were characterized in mammary tissue collected from lactating sows at slaughter. Tissue explants were incubated in an isosmotic physiologic buffer with [³H]taurine tracer to measure taurine uptake. Taurine uptake was dependent upon the presence of extracellular sodium and chloride ions, which is consistent with the co-transport of sodium and chloride with taurine. Uptake was not dependent upon ion exchange mechanisms or upon furosemide-sensitive ion co-transport. Taurine uptake was saturable and exhibited an apparent K_m of 20 μM and a V_max of 386 μmol/kg cell water/30 min. Substrate specificity studies indicated a strong interaction of β-amino acids with the taurine transport system. Taurine transport in lactating sow mammary tissue is therefore a high affinity, sodium-dependent mechanism specific for β-amino acids, and is analogous to sodium-dependent taurine uptake in other tissues. The high affinity and high specificity of the taurine uptake system allows for concentration of taurine within the mammary cell and is ultimately responsible for provision of taurine required for neonatal development.     

Cloning and expression of bovine imc-415 cDNA from mammary gland

Bovine imc-415 cDNA was cloned from mammary gland using RACE PCR; it coded for 245 amino acids. The deduced amino acid sequence of mouse and human showed about 94% identity. Expression of bovine imc-415 increased about 40% in involuted mammary tissues compared with lactating tissues.     

Cloning and Expression of Bovine imc-415 cDNA from Mammary Gland

Bovine imc-415 cDNA was cloned from mammary gland using RACE PCR; it coded for 245 amino acids. The deduced amino acid sequence of mouse and human showed about 94% identity. Expression of bovine imc-415 increased about 40% in involuted mammary tissues compared with lactating tissues.     

Tissue-specific effects of starvation and refeeding on the disposal of oral [1-14C]triolein in the rat during lactation and on removal of litter.

1. The effects of starvation and refeeding on the disposal of oral [14C]triolein between 14CO2 production and 14C-lipid accumulation in tissues of virgin rats, lactating rats and lactating rats with pups removed were studied. 2. Starvation (24 h) increased 14CO2 production in lactating rats and lactating rats with pups removed to values found in virgin rats. This increase was accompanied by decreases in 14C-lipid accumulation in mammary gland and pups of lactating rats and in white and brown adipose tissue of lactating rats with pups removed. 3. Short-term (2 h) refeeding ad libitum decreased 14CO2 production in lactating rats and lactating rats with pups removed, and restored the 14C-lipid accumulation in mammary glands plus pups and in white and brown adipose tissue respectively 4. Insulin deficiency induced with mannoheptulose inhibited the restoration of 14C-lipid accumulation in white adipose tissue on refeeding of lactating rats with pups removed, but did not prevent the restoration of 14C-lipid accumulation in mammary gland. 5. Changes in the activity of lipoprotein lipase in mammary gland and white adipose tissue paralleled the changes in 14C-lipid accumulation in these tissues. 6. It is concluded that 14C-lipid accumulation in mammary gland may not be affected by changes in plasma insulin concentration and that it is less sensitive to starvation than is lipogenesis or lactose synthesis. This has the advantage that the milk lipid content can still be maintained from hepatic very-low-density lipoprotein for a period after withdrawal of food. The major determinant of the disposal of oral 14C-triolein appears to be the total tissue activity of lipoprotein lipase. When this is high in mammary gland (fed lactating rats) or white adipose tissue (fed lactating rats with pups removed), less triacylglycerol is available for the muscle mass and consequently less is oxidized.     

The pattern of fatty acid synthesis in lactating rabbit mammary gland studied in vivo

The biosynthesis of fatty acids has been studied in lactating rabbits at 6h after intravenous injection of sodium [1-(14)C]acetate. The specific radioactivities of the individual fatty acids (C(6:0) to C(14:0)) and the proportions of these fatty acids synthesized were similar in mammary tissue and milk. Hexanoic acid had the highest specific radioactivity, and the C(8:0)-C(14:0) fatty acids had similar specific radioactivities, which were about five times those of C(16) and C(18) acids. No radioactivity was detected in fatty acids of chain length C(14) in these tissues were similar to those of the long-chain fatty acids in the milk and mammary gland. The results show that the C(4:0)-C(14:0) fatty acids are synthesized within the mammary gland rather than by fatty acid uptake from circulating blood or by oxidation of long-chain fatty acids within the gland. We conclude that de novo synthesis of esterified fatty acids in vivo by this tissue has a high degree of chain-length specificity.     

Effect of premature weaning on amino acid uptake by the mammary gland of lactating rats.

1. Arteriovenous differences of amino acids across the lactating mammary gland were measured in normal rats and weaned for 4, 5 and 24h. 2. Uptake of amino acids by mammary glands of rats weaned for 5h or more was significantly lower than that of controls. This was not reversed by injection of prolactin. 3. By using 'unilaterally weaned' rats we showed that milk accumulation plays an important role in amino acid uptake by mammary gland. 4. gamma-Glutamyltransferase activity was significantly lower in 'weaned' glands than in 'normal' glands. This provides further support for the hypothesis of the function of the gamma-glutamyl cycle in the mammary gland in vivo.